Our long term goal is to understand how the corneal epithelium maintains its barrier function against noxious agents and infection. We found that endothelin, ET-1 and epidermal growth factor, EGF, interact to control corneal epithelial proliferation and differentiation. The current project is directed towards obtaining a better understanding how these cytokines mediate their control of these responses. We hypothesize that their combined effects may stem from interactions between their signaling pathways which involve crosstalk and negative feedback. Our evidence for crosstalk and negative feedback is based on our findings that: 1. The mitogenic response to EGF is enhanced through crosstalk in the presence of ET-1; 2. The mitogenic response to EGF can be enhanced through inhibition of a negative feedback mechanism whereby EGF mediated increases in prostaglandin biosynthesis and protein kinase A activity suppress proliferation. The involvement of crosstalk will be evaluated based on a determination of whether the time dependence and magnitude of mitogen activated protein kinase (MAPK) activation induced by EGF alone are affected by the addition of ET-1. Based on evidence in some other tissues that EGF mediated increases in cAMP can stimulate differentiation through inhibition of Raf activation, we will test the hypothesis that increases in cAMP blunt and delay the activation of MAPKs and or pRb phosphorylation activation at the level of cell cycle control. We have other evidence that changes in membrane ion transport and cell volume are components of the cell signaling pathways linked to ET and EGF receptor stimulation. Accordingly, the third aim seeks to further characterize the role of such changes in linking ET and EGF receptor stimulation to the control of proliferation and differentiation. This pursuit can provide insight into whether crosstalk or negative feedback may also stem from the effects of ET-1 and EGF on one or both of these parameters. This will be determined based on measurements of the effects of ET-1 and EGF on membrane ion transport, cell volume, intracellular ionic activities and membrane voltage. Our goals will further determine whether interactions among components of the MAPK cascade together with changes in membrane ion transport and volume explain how ET enhances the mitogenic response to EGF. Furthermore these studies will shed light on how EGF receptor stimulation induces an autocrine mediated negative feedback which suppresses the mitogenic response to EGF. These studies could help identify strategies for use in the clinical setting to mitigate problems associated with recurrent corneal epithelial erosion.